This project aims to discover essential features in the mechanism of the sodium and potassium ion pump in the plasma membranes of animal cells by investigation of the sodium- and potassium-dependent adenosine triphosphatase purified from mammalian kidney. One approach compares this ATPase with 2 proton transport ATPases and with an uncharacterized ATPase from mycoplasma. The proton transport ATPases are from mammalian gastric mucosa and a bread mold, Neurospora; they will be supplied by collaboration with other laboratories. The comparison will be by fingerprinting radioactive phosphopeptides overlapping the active site of the phosphoenzymes. The phosphopepties will be isolated by proteolytic digestion and high-voltage paper electrophoresis. Corresponding properties of the peptides will relate transport ATPases over a much wider range of tissues and organisms than has been suspected. This relatedness will enlarge the family of transport ATPases. A second approach aims to characterize the homogeneity of Jorgensen's zonal preparation, one of the purest available. Since the ratio of ligand binding sites for ATP, covalent phosphate, vanadate, or ouabain to protomer peptide chains is about 1:2, the protomers are not homogeneous when liganded. This may reflect a regulatory control mechanism, subunit interaction, or merely partial denaturation. In order to investiagate this inhomogeneity, the response of the enzyme to potentially regulatory phosphorylating and dephosphorylating enzymes will be tested. Such enzymes will inclue various phosphoprotein phosphatases and phosphoprotein kinases. This pump generates concentration gradients of sodium and potassium ions, which in turn energize cell volume control and electrical or secretory activity such as that of brain or kidney. In the heart the pump is the receptor for drugs which counteract congestive heart failure.